For the past 30 years, tamoxifen (TAM) has been the most often prescribed endocrine treatment for breast cancer. In the past few years it was demonstrated that aromatase inhibitors are superior to TAM as adjuvant therapy in the postmenopausal patient population. Furthermore, aromatase inhibitors are effective as a second-line treatment following the emergence of TAM resistance and are being tested in the chemoprevention setting. This will bring a paradigm shift in the standard endocrine therapy for breast cancer in the near future. Identification of the key factors involved in the molecular mechanism of resistance to both TAM and aromatase inhibitors will undoubtedly lead to the development of logical therapeutic targets. We have developed and characterized a preclinical model of TAM resistance in the hormone-dependent T47D:A18 cell line that was engineered to overexpress protein kinase C alpha (PKCa). Tumors derived from these cells grow in athymic mice in an estrogen (E2)-independent and TAM-resistant fashion. Based on this model we examined clinical specimens and discovered that overexpression of PKCa is frequently associated with TAM resistance in human breast cancers. More recent preliminary data indicate that the T47D:A18/PKCa cells and tumors express high levels of Notch-4, a known breast oncogene and putative marker of breast cancer stem cells. Most interesting is our finding that T47D:A18/PKCa TAM-resistant tumors regress in the presence of E2 or raloxifene (RAL). It is our hypothesis that PKCa overexpressing, TAM-resistant tumors in patients may respond to treatment with RAL, E2 or Notch inhibitors. The T47D:A18/PKCa breast cancer tumor model will be used to investigate three therapeutic treatment strategies: (1) the opposing actions of TAM and RAL and the potential therapeutic application of RAL and other SERMS, (2) the role of Notch4 and effect of Notch inhibitors in TAM-resistance;and (3) the efficacy of estrogen as compared to aromatase inhibitor therapy. Relevance: The emergence of tamoxifen-resistant breast cancer is a critical problem in the management of advanced disease. Since essentially all patients with metastatic disease will relapse during tamoxifen treatment, alternative therapeutic strategies are needed. This proposal will address three potential approaches to treatment based on protein kinase C alpha (PKCa) as a biomarker to guide therapeutic choice.